Mechanical Effects of Aspirin on Clot Formation
Mitchell J George, Kevin R Aroom, Charles E Wade, Naveed U Saqib, Gustavo S Oderich, Charles S Cox, Jr., Brijesh S Gill
McGovern Medical School, Houston, TX
BackgroundAspirin is an anti-platelet drug taken by millions of patients to reduce risk of disease like stroke, myocardial infarction, or peripheral vascular disease. Aspirin exerts its antithrombotic effects by irreversibly inhibiting cyclooxygenase 1 & 2 (COX-1 & COX-2) and decreasing the conversion of arachidonic acid to thromboxane A2 by platelets. The inhibitory pathway of aspirin leading to decreased platelet aggregation has been understood since the 1970’s. However, the mechanical effects of aspirin on clot formation are unknown. There is no established mechanism of action of aspirin that involves the mechanical process of clot formation.
Therefore, this study aims to investigate the effects of aspirin on the mechanical process of clot formation in a gain and loss of function manner. We use a microelectromechanical systems (MEMS) based clot contraction sensor (CCS) within a warmed cartridge to measure the force of platelet contraction in fresh blood samples from healthy volunteers. We target the thromboxane A2 pathway by using in-vitro arachidonic acid and or 325 mg of ingested aspirin added in a gain and loss of function manner, respectively. We hypothesize that arachidonic acid, a platelet activator, will increase platelet contraction and that aspirin, an antiplatelet drug, will decrease platelet contraction.
MethodsVenous blood samples were taken from 100 consenting healthy adult volunteers before and ninety minutes after oral ingestion of 325 mg non-enteric coated aspirin tablets. Whole blood was either run immediately in a platelet contraction assay or stored in 3.2% citrate tubes for testing in an optical aggregometer. We target the TXA2 platelet pathway and measure changes in platelet contraction due to inhibition with aspirin or stimulation with arachidonic acid using a MEMS based clot contraction sensor (CCS).
Human SubjectsThis study was performed at University of Texas Health Science Center within the Texas Medical Center, Houston Texas, USA. Institutional Review Board approval was obtained prior to initiating the study (HSC-MS-16-0981). One hundred healthy human subjects above the age of eighteen who had abstained from taking any medication the previous two weeks were voluntarily enrolled. 50 males and 50 females were enrolled.
Blood Sample CollectionBlood samples were collected immediately before and ninety minutes after administration of 325 mg of oral non-enteric coated Aspirin. Blood was obtained via venipuncture in the antecubital fossa with a 20-ga butterfly needle into a sterile polypropylene syringe under good clinical practices. This fresh whole blood sample with no anticoagulation was either: 1) run immediately in the platelet contraction device, 2) transferred to serum separator tubes for measurement of prostanoids and fibrinogen or 3) anticoagulated with 3.2% citrate to measure platelet aggregation, complete blood count, and thromboelastrography.
Measuring Clot Contraction ForceClot contraction force was measured using a previously described MEMS based clot contraction sensor (CCS). Briefly, the CCS is a silicon chip with beam springs fabricated using deep reactive ion etching (Figure 1A). The CCS cartridge is assembled from seven components including a blood sample (Figure 1B). The cartridge resides in a heated chamber maintained at 37 degrees Celsius (not shown). A study sample of citrated and recalcified blood (7) is injected between two 6 mm diameter acrylic plates (1&6) and allowed to clot. The plates are 1 mm apart and the study sample is 35 micro-liters. As the study sample clots, the platelets within the sample contract and translate the superior acrylic plate (6) downward. The superior acrylic plate is attached to the sensor platform of the CCS (5). The CCS sensor platform (5) is attached to the sensor frame (3) by four beam springs. The sensor frame (3) of the CCS is fixed to the device platform (2). A camera with a microscope objective focuses on a tracking prism (4) to determine the downward displacement of the superior acrylic plate in micrometers. An orthogonal view of the entire device assembly is shown in Figure 6C and a cross-sectional view through the CCS cartridge is shown in Figure 6D.Force generated by the clot with time is derived using Hooke’s law. Whole blood, PRP or platelet suspensions are suitable for testing in this device. In the present study whole blood without anticoagulation from each sample was tested within four minutes of collection. Four conditions were measured: 1) pre-Aspirin, 2) pre-Aspirin with in-vitro AA added, 3) post-Aspirin, and 4) post-Aspirin with in-vitro AA added. Assays required 250 µL of blood and lasted 80 minutes. 10 µL of 50 mMol arachidonic acid (AA) for. Metrics reflecting platelet contraction
taken from resultant force curves include contraction start time (CST) and maximum contraction force (MCF). Assays were run in duplicate.
Optical Aggregometry Optical aggregometry was performed using a Chronolog Model 700 Whole Blood Optical Aggregometer (Chrono-log Corp., Havertown, PA). Maximum amplitude (MA), slope, lag time and area under the curve (AUC) was recorded. Assays were run in triplicate.
Thromboxane A2 LevelsSerum thromboxane A2 (TXA2) was measured indirectly by measuring its stable metabolite thromboxane B2 (TXB2 with commercially available ELISA kits (Enzo Life Sciences, Farmingdale, NY, USA). Measurements were run in triplicate.
Fibrinogen Levels from Serum Fibrinogen was measured in serum from serum separator tubes with commercially available ELISA kits (Enzo Life Sciences, Farmingdale, NY, USA). Measurements were run in triplicate.
Statistical AnalysisData from platelet contraction assays were compared using a one-way repeated measures analysis of variance (ANOVA). Post-hoc analysis with Bonferroni pairwise comparison of
means measured differences among the four different conditions. Paired t-tests compared data from before and after taking aspirin for individual conditions. Data is reported as median and interquartile range (IQR).
ResultsAspirin Reduces Production of TXB2 and Aggregation in Platelets
TXB2 was measured from serum in volunteer samples before and after ingestion of a 325 milligram enteric coated aspirin (Figure 2). Levels of TXB2 were widely variable and are presented on a logarithmic scale. Median and interquartile range of serum TXB2 levels prior to aspirin ingestion were 11,135 (5,470, 26,765) pg/mL and after aspirin ingestion decreased to 705 (400, 1,234) pg/mL. The average drop in TXB2 was 9,979 pg/mL. This drop in TXB2 levels was expected and reflective of aspirin’s inhibition of COX-1 and COX-2. Similarly, platelet aggregation was measured from platelet rich plasma before and after ingestion of a 325 milligram enteric coated aspirin using a Chronolog Model 700. Percent aggregation prior to aspirin ingestion was 71 (41, 100) and after aspirin ingestion fell to 1 (0, 11).
Clot Contraction Force Increases after Aspirin IngestionUnexpectedly, clot contraction forces increased after aspirin ingestion (Figure 3a). Using a paired t-test, there was a significant difference between inter-individual force values with p = 0.001. Comparing males to females, this effect on clot contraction from aspirin was only seen in males. The difference in females was not statistically significant.
Arachidonic Acid Increases Clot Contraction Force Before and After Aspirin IngestionArachidonic acid increased the force of clot contraction compared to controls (Figure 3b). Using a paired t-test, there was a significant difference between these two groups with p = 0.0001. Unexpectedly, this increase in force with arachidonic acid treatment was sustained even after aspirin ingestion (Figure 3c).Clot Fibrin Concentration Decreases After Aspirin IngestionFibrinogen from serum was measured before and after aspirin ingestion. The level of fibrinogen after aspirin ingestion in serum was increased, implying that fibrin content of clots formed after aspirin ingestion was decreased.
ConclusionsIn this study, we demonstrate the effects of aspirin on platelet contraction through gain and loss of function experiments. Healthy subjects were administered 325 mg of enteric coated oral aspirin. Inhibition of platelet TXA2 production through the COX pathway was confirmed by measuring optical aggregometry and levels of TXB2. Blood samples drawn before and after aspirin administration were tested in a clot contraction device and unexpectedly, contraction forces increased after aspirin ingestion. This increase in platelet contraction force after aspirin ingestion was only observed in males. In addition, the increase in clot contraction forces from the addition of arachidonic acid were maintained after aspirin ingestion.
The decrease in fibrin content of forming clots is a potential explanation for the increase in clot contraction after aspirin ingestion. One potential mechanism of this phenomena is acetylation of lysin residues on fibrin by aspirin. The findings of increased clot contraction after aspirin ingestion is important clinically because it demonstrates an additional beneficial
mechanism of aspirin in thrombotic disease like stroke, myocardial infarction or venous thromboembolism. Aspirin encourages softer clots and thus increases recanalization of vessels after a thrombotic event.
Figure 1. The clot contraction sensor (A) sits on top of the sensor cartridge (B). The cartridge sits within a heated aluminum block and is imaged by a camera (C&D).
Figure 2. Maximum platelet contraction force significantly increases after aspirin ingestion compared to controls (a), and after addition of arachidonic acid (b). Increases in platelet contraction forces are sustained after arachidonic acid agonism even after aspirin ingestion.
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